This invention relates generally to methods of detecting the presence of oxidatively damaged proteins and endogenous/autoantibodies to oxidatively damaged proteins.
Chronic inflammation and oxidative stress are associated with a wide variety of diseases and disorders in human populations. Such diseases and disorders affect organs and systems including, but are not limited to, reproductive organs, immune system, lungs, cardiovascular system, nervous system, gastrointestinal system, as well as organs and systems controlling growth and development. Such diseases include, but are not limited to, coronary artery disease, renal disease, cancer, and psychiatric diseases.
Inflammation and oxidative stress in animals result from interaction with the environment and involve exposure to a wide variety of physical, chemical and biological agents (Ames, B. N. et al. (1993), “Oxidants, antioxidants, and the degenerative diseases of aging,” Proc. Natl. Acad. Sci. USA 90:7915-7922; Sies, H. (ed.) (1991), Oxidative Stress, Oxidants and Antioxidants, New York: Academic Press). When these conditions become chronic, they can lead to changes in the normal cellular balance between antioxidants and oxidant that are associated with many different diseases in aging human populations (Grisham, M. G. (1994), “Oxidants and free radicals in inflammatory bowel disease,” Lancet 344:859-861; Halliwell, B. (1996), “Antioxidants in human health and disease,” Annu. Rev. Nutr. 16:33-50; Jenner, P. (1994), “Oxidative damage in neurodegenerative disease,” Lancet 344:796-798; Sohal, R. S. and Weindruch, R. (1996), “Oxidative stress, caloric restriction, and aging,” Science 273:59-63; Weitzman, S. A. and Gordon, L. I. (1990), “Infla mation and cancer: role of phagocyte-generated oxidants in carcinogenesis,” Blood 76:655-663; Winterboum, C. C. (1995), “Nutritional antioxidants: their role in disease prevention,” New Zealand Med. J. 108:447-449). For example, there is increasing evidence that atherosclerosis is a chronic inflammatory disease that develops in response to metabolic, physical or environmental injuries such as hypercholesterolemia, hypertension or cigarette smoking (Munro, J M and Cotran, R. S. (1988), “The pathogenesis of atherosclerosis: Atherogenesis and inflammation,” Lab. Invest. 58:249-261; Parthasarathy, S. et al. (1992), “The role of oxidized low-density lipoproteins in tie pathogenesis of atherosclerosis,” Annu. Rev. Med. 43:219-225; Reaven, P. D. and Witztum, J. L. (1996), “Oxidized low density lipoproteins in atherogenesis: Role of dietary modification,” Annu. Rev. Nutr. 16:51-71; Ross, R. (1993), “The pathogenesis of atherosclerosis: a perspective for the 1990s,” Nature 362:801-809). Development of atherosclerosis is associated with many cardiovascular complications involving organs such as the heart, kidney and brain (Maggi, E. et al. (1994) Kidney Intl. 45:876-883).
Sulfur moieties in amino acid residues in proteins are particularly susceptible to oxidation. For example, cysteine residues (which contain a thiol moiety) and cystine residues (which contain a disulfide moiety) can be oxidized to cysteic acid (cysteine sulfonic acid) and molecules with other oxidation states, depending on conditions. Likewise, the selenium analogs of cysteine and cystine, selenocysteine and selenocystine, which are more easily oxidized than their sulfur analogs, can be oxidized to selenocysteic acid (Huber, R. and Criddle, R. (1967), “Comparison of the Chemical Properties of Selenocysteine and Selenocystine with Their Sulfur Analogs,” Arch. Biochem. Biophys. 122:164-173). The sulfur moiety in methionine residues in proteins can be oxidized to the sulfoxide and, under stronger conditions to the sulfone. The oxidation of proteins in wool by performic or peracetic acids has been described (Maclaren, J. A. et al. (1959), “The oxidation of disulphide groups in proteins,” Biochim. Biophys. Acta 35: 280-281). The oxidation of hair proteins from bleaching has been studied, and the main site of degradation is thought to occur at the disulfide bonds of the cystinyl residues in the fibers (Robbins, C. R. (1988) Chemical and Physical Behavior of Human Hair, 2d ed. pp. 102-121). Selenomethionine is oxidized to the selenone, but to Applicants' knowledge, this has never been studied in proteins. Burk, R. and Mill, K. (1993), “Regulation of Selenoproteins,” Annu. Rev. Nutr. 13:65-81, provide a review of selenium in proteins.
Reagents capable of oxidizing sulfur or selenium moieties in sulfur- or selenium-containing amino acids in proteins to sulfonic or selenocysteic acid moieties and sulfone or selenone moieties may be encountered directly in the environment (e.g., ozone), or may be generated endogenously, e.g., hypochlorous acid (HOCl) is generated by the myeloperoxidase (MPO)/hydrogen peroxide (H2O2)/chloride ion (Cl−) system of activated phagocytic leukocytes during inflammation.
A number of exogenous sources of strong oxidants exist that are potentially important in chronic human exposures These reportedly include ozone, radiation, chlorination processes that give rise to chloramines, oxides of nitrogen, iron and copper salts that promote oxidizing radical formation via Fenton chemistry, and normal dietary phenolic compounds (e.g., caffeic acid) that generate oxidants by redox cycling (Ames, B. N. et al. (1993), “Oxidants, antioxidants, and the degenerative diseases of aging,” Proc. Natl. Acad. Sci. USA 90:7915-7922; Berlett, B. S. et al. (1996), “Comparison of the effects of ozone on the modification of amino acid residues in glutamine synthetase and bovine serum albumin,” J. Biol. Chem. 271:4177-4182; Sies, H.,(ed.) (1991), Oxidative Stress, Oxidants and Antioxidants, New York: Academic Press; Stadtman, E. R. (1995), “Role of oxidized amino acids in protein breakdown and stability,” Meth. Enzymol. 258:379-393; Thomas, E. L. et al. (1986), “Preparation and characterization of chloramines,” Meth. Enzymol. 132:569-585; Fliss, H. and Menard, M. (1994), “Rapid neutrophil accumulation and protein oxidation in irradiated rat lungs,” J. Appl. Physiol. 77:2727-2733). Thus, interaction with a wide variety of environmental oxidants may also contribute to oxidative stress in vivo and the formation of oxidized sulfur or selenium moieties in sulfur- or selenin-containing amino acids, e.g., cysteic acid in proteins.
In addition, endogenous sources of strong oxidants include but are not limited to aerobic mitochondrial respiration, peroxisomes, and cytochrome P450 enzymes (Ames, B. N. et al. (1993), “Oxidants, antioxidants, and the degenerative diseases of aging,” Proc. Natl. Acai Sci. USA 90:7915-7922; Sies, H. (ed.) (1991), Oxidative Stress, Oxidants and Antioxidants, New York: Academic Press).
Another major endogenous source of oxidative stress derives from the involvement of phagocytic leukocytes (neutrophils, monocytes/macrophage, eosinophils), which function in defense against environmental and endogenous agents (Jesaitis, A. J. and Dratz, E. A. (eds.) (1992) The Molecular Basis of Oxidative Damage by Leukocytes, CRC Press, Boca Raton; Klebanoff, S. J. and Clark, R. A. (1978), The Neutrophil: Function and Clinical Disorders, North Holland, Amsterdam; Smith, J. A. (1994), “Neutrophils, host defense, and inflammation: a double-edged sword,” J. Leukoc. Biol. 56:672-686). A major feature of this host defense function is a powerful oxygen-dependent, microbicidal, viricidal and tumoricidal system that utilizes two different peroxidases, mycloperoxidase (MPO) and eosinophilic peroxidase (EPO) (Henderson, W. R., Jr. (1991) “Eosinophil peroxidase: occurrence and biological function,” in Peroxidases in Chemistry and Biology, Vol 1, Everse, J. el al. (eds.), CRC Press, Boca Raton, pp. 105-121; Klebanoff, S. J. (1992), “Oxygen metabolites from phagocytes,” in Inflammation. Basic Principles and Clinical Correlates, J. I. Gallin et al. (eds.), Raven Press, NY, pp. 391-444). MPO is found only in the granules of neutrophils and monocytes/macrophages, and is biochemically distinct from EPO (Bainton, D. F. (1992), “Developmental biology of neutrophils and eosinophils,” in Inflammation: Basic Principles and Clinical Correlates, 2nd ed., Gallin, J. L. et al. (eds.), Raven Press, NY, pp. 303-324; Henderson, W. R., Jr. (1991) “Eosinophil peroxidase: occurrence and biological function,” in Peroxidases in Chemistry and Biology, Vol 1, Everse, J. et al. (eds.), CRC Press, Boca Raton, pp. 105-12; Nichols, B. A. and Bainton, D. F. (1973), “Differentiation of human monocytes in bone marrow and blood. Sequential formation of two granule populations,” Lab. Invest. 29:27-40). Interaction of these cells with a variety of soluble and nonsoluble agonists leads to a respirator burst (Gallin, J. I. et al. (eds.) (1992) Inflammation: Basic Principles and Clinical Correlates, Second Edition, Raven Press, Ltd., New York) and activation of an NADPH-dependent oxidase complex that generates large quantities of superoxide radical anion, a substantial portion of which dismutates to hydrogen peroxide (H2O2) (Segal, A. W. and Abo, A. (1993), “The biochemical basis of the NADPH oxidase of phagocytes,” Trends Biochem. Sci. 18:43-47). In this process, cytoplasmic granules are mobilized and undergo secretion into both intracellular and extracellular spaces (Dahlgren, C. et al. (1989), “Localization of the luminol-dependent chemi-luminescence reaction in human granulocytes,” J. Bioluminescence Chemiluminescence 4:263-266; Edwards, S. W. (1987), “Luminol- and lucigenin-dependent chemiluminescence of neutrophils: role of degranulation,” J. Clin. Lab. Immunol. 22:35-39).
MPO utilizes H2O2 and Cl− (Carr, A. C. et al. (1996). “Peroxidase-mediated bromination of unsaturated fatty acids to form bromohydrins,” Arch. Biochem. Biophys. 327:227-233; Thomas, E. L. and Learn, D. B. (1991), “Myeloperoxidase-catalyzed oxidation of chloride and other halides: The role ofchloramines,” in Peroxidases in Chemistry and Biology, Vol 1, Everse, J. et al. (eds.), CRC Press, Boca Raton, pp. 83-103) to catalyze the formation of the powerful oxidizing and halogenating species, hypochlorous acid (HOCl) (Harrison, J. E. and Schultz, J. (1976), “Studies on the chlorinating activity of myeloperoxidase,” J. Biol. Chem. 251:1371-1374; Klebanoff, S. J. (1992), “Oxygen metabolites from phagocytes,” in Inflammation. Basic Principles and Clinical Correlates, J. I. Gallin et al. (eds.), Raven Press, NY, pp. 391-144), (pKa=7.5, so at physiological pH one has both OCl− and HOCl) (Morris, J. C. (1966), “The acid ionization constant of HOCl from 5 to 35°,” J. Phys. Chem. 70:3798-3805). The MPO-H2O2—Cl− system of activated neutrophils (1-5×106) is reported to be able to produce 100-200 nanomoles of HOCl in two hours (Kalyanaraman, B. and Sohmle, P. G. (1985), “Generation of free radical intermediates from foreign compounds by neutrophil-derived oxidants,” J. Clin. Invest. 75:1618-1622; Weiss, S. J. (1989), “Tissue destruction by neutrophils,” N. Engl. J. Med. 320:365-376). In large interstitial inflammatory sites, the concentration of HOCl has been estimated to be in the mM range (Weiss, S. J. (1989), “Tissue destruction by neutrophils,” N. Engl. J. Med. 320:365-376). Reactivity of the MPO-H2O2—Cl− system is enhanced when phagocytes are activated on biological surfaces (Nathan, C. F. (1987), “Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes,” J. Clin. Invest. 80:1550-1560), and when the highly cationic MPO binds to cell surfaces or anionic macromolecules (Britigan, B. E. et al. (1996), “Binding of myeloperoxidase to bacteria: effect on hydroxylradical formation and susceptibility to oxidant-mediated killing,” Biochim. Biophys. Acta 1290:231-240; Olszowska, E. et al. (1989), “Enhancement of proteinase-mediated degradation of proteins modified by chlorination,” Int. J. Biochem. 21:799-805).
In mammalian cells, EPO also is able to catalyze the formation of HOCl, however, its preferred in vivo substrate is thought to be bromide and/or thiocyanate, both of which give rise to potent oxidants (HOBr and HOCN, respectively) (Can, A. C. et al. (1996), “Peroxidase-mediated bromination of unsaturated fatty acids to form. bromohydrins,” Arch Biochem. Biophys. 327:227-233; Thomas, E. L. et al. (1995), “Oxidation of bromide by human leukocyte enzymes myeloperoxidase and eosinophil peroxidase. Formation of bromamines,” J. Biol. Chem. 270:2906-2913). HOCl can also react with superoxide radical anion in a metal-ion independent Haber-Weiss type reaction to form hydroxyl radical (−OH) or with H2O2 to form singlet oxygen (1O2) (Candeias, L. P et al. (1993), “Free hydroxyl radicals are formed on reaction between the neutrophil derived species superoxide anion and hypochlorous acid,” FEBS Lett. 333:151-153). Recent evidence suggests that Haber-Weiss chemistry also involves production of 1O2 (Khan, A. U. and Kasha, M. (1994), “Singlet molecular oxygen in the Haber-Weiss reaction,” Proc. Natl. Acad. Sci. USA 91:12365-12367).
Under acidic conditions in the presence of chloride ion, HOCl is in equilibrium with chlorine gas, and phagocytes have been shown to utilize this powerful oxidant at sites of inflammation and vascular disease (Hazen, S. L. et al. (1996), “Human neutrophils employ chlorine gas as an oxidant during phagocytosis,” J. Clin. Invest. 98:1283-1289). Both phagocytic and endothelial cells produce nitric oxide (NO) and reaction of NO with superoxide leads to formation of peroxynitrite (Halliwell, B. (1996), “Antioxidants in human health and disease,” Annu. Rev. Nutr. 16:33-50), a strong oxidant with properties similar to hydroxyl radical, which itself, can be formed by homolysis of peroxynitrite (Floris, R. et al. (1993), “Interaction of myeloperoxidase with peroxynitrite. A comparison with lactoperoxidase, horseradish peroxidase and catalase,” Eur. J. Biochem. 215:767-775). In principle, all of these reactive oxygen species (ROS) and reactive non-oxygen species are strong enough to oxidize the sulfur or selenium moieties of sulfur- or selenium-containing amino acid residues in proteins to cysteic acid (cysteine sulfonic acid) or selenocysteic acid and to sulfone or selenone moieties such as methionine sulfone or selenone.
In vitro studies with model compounds show that HOCl reacts at least 100 times faster with thiols compared to primary amines (Folkes, L. K. et al. (1995), “Kinetics and mechanisms of hypochlorous acid reactions,” Arch. Biochem. Biophys. 323:120-126; Winterboum, C. C. (1985), “Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide-chloride, and similarity of the oxidant to hypochlorite,” Biochim. Biophys. Acta 840:204-210). In the presence ofexcess thiol, HOCl cxidation leads to disulfide formation (Silverstein, R. M. and Hager, L. P. (1974), “The chloroperoxidase—catalyzed oxidation of thiols and disulfides to sulfenyt chlorides,” Biochemistry 13:5069-5073). However, in the absence of excess thiol (a condition that exists in vivo at sites of inflammation) (Fliss, H. and Ménard, M. (1994), “Rapid neutrophil accumulation and protein oxidation in irradiated rat lungs,” J. Appl. Physiol. 77:2727-2733) the disulfide may be further oxidized, through a sulfenyl chloride, to the sulfonic acid (Drozdz, R. et al. (1988), “Oxidation of amino acids and peptides in reaction with myeloperoxidase, chloride and hydrogen peroxide,” Biochem. Biophys. Acta 957:47-52; Pereira, W. E. et al. (1973), “Chlorination studies, II. The reaction of aqueous hypochlorous acid with α-amino acids and dipeptides,” Biochim. Biophys. Acta 313:170-180; Silverstein, R. M. and Hager, L. P. (1974), “The chloroperoxidase—catalyzed oxidation of thiols and disulfides to sulfonyl chlorides,” Biochemistry 13:5069-5073; Little, C. and O'Brien, P. (1967), “Products of Oxidation of a Protein Thiol Group after Reaction with Various Oxidizing Agents,” Arch. Biochem. Biophys. 122:406-410; Little, C. and O'Brien, P. J. (1969), “Mechanism of Peroxide-Inactivation of the Sulphydryl Enzyme Glyceraldehyde-3-Phosphate Dehydrogenase,” Eur. J. Biochem. 10:533-538; Coan, C. et al. (1992), “Protein Sulfhydryls are Protected from Irreversible Oxidation by Conversion to Mixed Disulfides,” Arch. Biochem. Biophys. 295:369-378).
It appears that most investigators have assumed that HOCl oxidation of cysteine, generally measured as loss of cysteine thiol, leads exclusively to the disulfide; further oxidation to sulfinic and sulfonic acids has rarely been considered (Thomas, E. L. (1979), “Myeloperoxidase, hydrogen peroxide, chloride antimicrobial system: nitrogen-chlorine derivatives of bacterial components in bactericidal action against Escherichia coli,” Infect. Immun. 23:522-531). In agreement with model studies, treatment of proteins with several oxidizing halogen reagents has been shown to produce sulfenyl halides (Aune, T. M. and Thomas, E. L. (1978), “Oxidation of protein sulfhydryls by products of peroxidase-catalyzed oxidation of thiocyanate ion,” Biochemistry 17:1005-1010; Cunningham, L. W. and Nuenke, B. J. (1960), “Analysis of modified β-lactoglobulins and ovalbumins prepared from the sulfenyl iodide intermediates,” J. Biol. Chem. 235:1711-1715; Glazer, A. N. (1970), “Specific chemical modification of proteins,” Annu. Rev. Biochem. 39:101-130; Thomas, E. L. (1979), “Myeloperoxidase, hydrogen peroxide, chloride antimicrobial system: nitrogen-chlorine derivatives of bacterial components in bactericidal action against Escherichia coli,” Infect. Immun. 23:522-531). Spontaneous oxidation (resulting from dissolved oxygen) of these reactive intermediates gives rise to cysteic acid moieties (Silverstein, R. M. and Hager, L. P. (1974), “The chloroperoxidase—catalyzed oxidation of thiols and disulfides to sulfenyl chlorides,” Biochemistry 13:5069-5073), particularly since many proteins are unable to form disulfide bonds because of stearic hindrance (Glazer, A. N. (1970), “Specific chemical modification of proteins,” Annu. Rev. Biochem. 39:101-130; Little, C. and O'Brien, P. (1967), “Products of Oxidation of a Protein Thiol Group after Reaction with Various Oxidizing Agents,” Arch. Biochem. Biophys. 122:406-410; Coan, C. et al. (1992), “Protein Sulfhydryls are Protected from Irreversible Oxidation by Conversion to Mixed Disulfides,” Arch. Biochem. Biophys. 295:369-378).
HOCl oxidation of serum albumin, both isolated and in plasma, showed that all protein sulfur was oxidized before any primary amines reacted (Arnhold, J. et al. 1990), “On the action of hypochlorite on human serum albumin,” Biomed. Biochim. Acta 49:991-997; Hu, M.-L. et al (1993), “Antioxidant protection against hypochlorous acid in human plasma,” J. Lab. Clin. Med. 121:257-262). The stoichiometry indicated that oxidation beyond the disulfide occurred, and it was suggested that albumin (the main contributor to a plasma thiol concentration of 469 μM) serves as a major antioxidant defense against HOCl oxidation in vivo (Hu, M.-L. et al (1993), “Antioxidant protection against hypochlorous acid in human plasma,” J. Lab. Clin. Med. 121:257-262).
HOCl oxidation of low density lipoprotein (LDL) was reported to result in loss of all thiols in the protein constituent, apolipoprotein B-100, (apoB) at a concentration of HOCl that formed little, if any, chloramines with the many primary amino groups of lysine side chains or oxidation of lipid (Amhold, J. et al. (1991), “Modification of low density lipoproteins by sodium hypochlorite,” Biomed. Biochim. Acta 8:967-973; Hazell, L. J. and Stocker, R. (1993), “Oxidation of low-density lipoprotein with hypochlorite causes transformation of the lipoprotein into a high-uptake form for macrophages,” Biochem. J. 290:165-172; Hazell, L. J. et al. (1994), “Oxidation of low-density lipoprotein by hypochlorite causes aggregation that is mediated by modification of lysine residues rather than lipid oxidation,” Biochem. J. 302:297-304; Hu, M.-L. et al (1993), “Antioxidant protection against hypochlorous acid in human plasma,” J. Lab. Clin. Med. 121:257-262). HOCl oxidation of low density lipoproteins (LDL) appears to produce sulfinic acids (Yang, E. Y. et al (1999), “Selective modification of apoB-100 in the oxidation of low density lipoproteins by myeloperoxidase in vitro,” J. Lipid Res. 40:686-98). HOCl oxidation of immunoglobulins and several other proteins (Naskalski, J. W. (1994), “Oxidative modification of protein structures under the action of myeloperoxidase and the hydrogen peroxide and chloride system,” Ann. Biol. Clin. 52:451-456) was suggested to produce cysteic acid residues, but this was never directly demonstrated by identifying and assaying the suspected product (Hu, M.-L. et al (1993), “Antioxidant protection against hypochlorous acid in human plasma,” J. Lab. Clin. Med. 121:257-262; Naskalski, J. W. (1994), “Oxidative modification of protein structures under the action of myeloperoxidase and the hydrogen peroxide and chloride system,” Ann. Biol. Clin. 52:451-456; Thomas, E. L. (1979), “Myeloperoxidase, hydrogen peroxide, chloride antimicrobial system: nitrogen-chlorine derivatives of bacterial components in bactericidal action against Escherichia coli,” Infect. Immun. 23:522-531).
Bacterial killing by the MPO-H2O2—Cl− system has been shown to be directly related to loss of sulfhydryl groups, and the stoichiometry of the reaction suggests that some sulfonic acid may have been formed (Thomas, E. L. (1979), “Myeloperoxidase, hydrogen peroxide, chloride antimicrobial system: nitrogen-chlorine derivatives of bacterial components in bactericidal action against Escherichia coli,” Infect. Immun. 23:522-531). Oxidation of thiol groups in P338D1 murine tumor cells by low concentrations of HOCl (50-60 μM) led to the stoichiometric formation of disulfide, but at somewhat higher concentrations of HOCl (120-150 μM) the disulfides disappeared (Schraufstätter, I. U. et al. (1990), “Mechanisms of hypochlorite injury of target cells,” J. Clin. Invest. 85:554-562). Although the product(s) was not identified, it is reasonable to assume that protein cysteic acid residues were produced.
Oxidized sulfur-containing amino acid residues have been found to be associated with many diseases which affect mammals, particularly humans. In humans, cysteic acid has been reported to occur in proteins isolated from four sources known to be under oxidative stress: senile cataractous lens tissue (Garner, M. H. and Spector, A. (1980), “Selective oxidation of cysteine and methionine in normal and senile cataractous lenses,” Proc. Natl. Acad. Sci. USA 77:1274-1277), ethrocyte spectrin from diabetic subjects (Schwartz, R. S. et al. (1991), “Oxidation of spectrin and deformability defects in diabetic erythrocytes,” Diabetes 40:701-708), immunoglobulin G from inflammatory synovial fluid (Jasin, H. E. (1993), “Oxidative modification of inflammatory synovial fluid immunoglobulin G,” Inflammation 17:167-181), and some hair proteins (Zan, H. and Gattner, H. G. (1997), “Hair sulfur amino acid analysis,” EXS 78:239-258). Two groups of unidentified proteins (possibly lipoproteins and albumin (Witko-Sarsat, V. et al. (1996), “Advanced oxidation protein products as a novel marker of oxidative stress in uremia” Kidney Intl. 49:1304-1313) have been observed in the plasma of patients with renal disease that appear to be similar to proteins obtained by treatment of normal human plasma with HOCl (Witko-Sarsat, V. et al. (1996), “Advanced oxidation protein products as a novel marker of oxidative stress in uremia,” Kidney Intl. 49:1304-1313). A monoclonal antibody (mAb) has been produced against human HOCl-oxidized LDL (oxLDL) that cross-reacts with other HOCl-oxidized proteins but was reported to not cross-react with LDL modified with reactive aldehyde products of lipid peroxidation or LDL that has been oxidized with copper (Cu2+) (Malle, E. et al. (1995), “Immunologic detection and measurement of hypochlorite-modified LDL with specific monoclonal antibodies,” Arterioscler. Thromb. Vasc. Biol. 15:982-989). Immunohistochemical studies using this mAb demonstrated the presence of HOCl-oxidized proteins in human atherosclerotic lesions (Hazell, L. J. et al. (1996), “Presence of hypochlorite-modified proteins in human atherosclerotic lesions,” J. Clin. Invest. 97:1535-1544) and glomerulosclerotic lesions (Malle, E. et al. (1997), “Immunological evidence for hypochlorite-modified proteins in human kidney,” Am. J. Pathol. 150:603-615) and reactivity correlated with severity of disease (Hazell, L. J. et al. 1996), “Presence of hypochlorite-modified proteins in human atherosclerotic lesions,” J. Clin. Invest. 97:1535-1544). MPO has also been shown immunohistochemically to be present in these lesions (Daugherty, A. et al. (1994), “Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions,” J. Clin. Invest. 94:437-444; Hazell, L. J. et al. (1996), “Presence of hypochlorite-modified proteins in human atherosclerotic lesions,” J. Clin. Invest. 97:1535-1544; Malle, E. et al. (1997), “Immunological evidence for hypochlorite-modified proteins in human kidney,” Am. J. Pathol. 150:603-615). Taken together, these data strongly implicate MPO and HOCl in the modification of proteins in vivo.
Protein carbonyl derivatives are also reported to be formed from oxidative injury. A method of derivatizing carbonyl groups using 2,4-dinitrophenylhydrazine (DNPH) and probing with a commercial biotinylated anti-DNP antibody followed by reacting with a streptavidin-linked horseradish peroxidase has been reported (Buss, H. et al (1997), “Protein carbonyl measurement by a sensitive ELISA method,” Free Radic. Biol. Med. 23:361-366; Winterbourn. C. C. and Buss, I. H. (1999), “Protein Carbonyl Measurement by Enzyme-Linked Immunosorbent Assay,” Methods in Enzymology 300:106-111).
Cysteine (thiol) and cystine (disulfide) residues are generally less abundant in proteins than other amino acid side chains, but their importance in the structure and function of proteins is universally recognized. Oxidation or other chemical modification of protein thiols and disulfides usually leads to loss of biological activity (Albrich, J. M. et al. (1981), “Biological reactivity of hypochlorous acid: implications for microbicidal mechanisms of leukocyte myeloperoxidase,” Proc. Natl. Acad. Sci. USA 78:210-214; Little, C. and O'Brien, P. (1967), “Products of Oxidation of a Protein Thiol Group after Reaction with Various Oxidizing Agents,” Arch. Biochem. Biophys. 122:406-410; Little, C. and O'Brien, P. J. (1969), “Mechanism of Peroxide-Inactivation of the Sulphydryl Enzyme Glyceraldehyde-3-Phosphate Dehydrogenase,” Eur. J. Biochem. 10:533-538).
Protein cysteic acid (cysteine sulfonic acid) is the stable end-product of oxidation of the functional sulfur moieties of cysteine and cystine and is not a normal constituent of naturally occurring mammalian proteins (Manneberg, M. et al. (1995), “Oxidation of cysteine and methionine residues during acid hydrolysis of proteins in the presence of sodium azide,” Anal. Biochem. 224:122-127; Manneberg, M. et al. (1995), “(Quantification of cysteine residues following oxidation to cysteic acid in the presence of sodium azide,” Anal. Biochem. 231:349-353). Depending on the oxidation conditions, a variety of intermediate oxidation states may be present (Maclaren, J. A. et al. (1959), “The oxidation of disulphide groups in proteins,” Biochim. Biophys. Acta 35: 280-281). To Applicants' knowledge, selenocysteic acid has never been identified in any natural protein, either in vivo or following oxidation in vitro. This most likely relates to the fact that selenocysteic acid is unstable under the conditions of acid hydrolysis employed in conventional amino acid analysis (Huber, R. and Criddle, R. (1967), “Comparison of the Chemical Properties of Selenocysteine and Selenocystine with Their Sulfur Analogs,” Arch. Biochem. Biophys. 122:164-173). There is no evidence that protein cysteic acid or selenocysteic acid undergoes further oxidation or reduction in vivo or alteration during storage in vitro.
Reduced selenium uptake has been associated with a number of clinical disorders in humans and animals, including cancer and heart disease (Arthur and Beckett, 1994), and appears to represent a form of oxidative stress (McLeod, R. et al. (1997), “Protection Conferred by Selenium Deficiency against Aflatoxin B1 in the Rat is Associated with the Hepatic Expression of an Aldo-Keto Reductase and a Glutathione S-Transferase Subunit That Metabolize the Mycotoxin,” Cancer Res. 57:4257-4266). Conversely, exogenously provided selenium acts as a chemopreventive agent, but the mechanism(s) through which these biological effects are mediated is not known. It has been suggested that the chemopreventive action of selenium involves its incorporation into selenoproteins (Gallegos, A. et al. (1997), “Mechanisms of the Regulation of Thioredoxin Reductase Activity in Cancer Cells by the Chemopreventive Agent Selenium,” Cancer Res. 57:4965-4970).
It is now recognized that selenocysteine is utilized in ribosome-mediated protein synthesis and its specific incorporation into protein is directed by the UGA codon (Burk, R. F. and Hill, K. E. (1993), “Regulation of Selenoproteins,” Annu. Rev. Nutr. 13:65-81; Stadtman, T. C. (1996), “Selenocysteine,” Annu. Rev. Biochem. 65:83-100). Selenocysteine is a functional residue in a number of enzymes that exhibit important antioxidant properties (Gallegos, A. et al. (1997), “Mechanisms of the Regulation of Thioredoxin Reductase Activity in Cancer Cells by the Chemopreventive Agent Selenium,” Cancer Res. 57:4965-4970): these include cellular and plasma glutathione peroxidases, phospholipid hydroperoxide glutathione peroxidase and thioredoxin reductase. Thioredoxin has been shown to regulate gene transcription by controlling the redox state of several transcription factors and their binding to DNA (Matthews, J. R. et al., 1992; Hirota, K. et al. (1997), “AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1.” Proc. Natl. Acad. Sci. USA 94:3633-3638). The thioredoxin gene is overexpressed in human cancer and inhibits apoptosis (Baker, A. et al. (1997), “Thioredoxin, a Gene Found Overexpressed in Human Cancer, Inhibits Apoptosis in Vitro and in Vivo,” Cancer Res. 57:5162-5167).
Interestingly, selenocysteine is also reported to be found in several deiodinases that act on thyroid hormones (Larsen and Berry (1995), “Nutritional and Hormonal Regulation of Thyriod Hormone Deiodinases,” Annu. Rev. Nutr. 15:323-352; Stadtman, T. C. (1996), “Selenocysteine,” Annu. Rev. Biochem. 65:83-100) substances that are involved in stimulating cellular oxidation.
Animals do not appear to distinguish methionine from selenomethionine (Burk and Hill, 1993), and selenomethionine can readily replace methionine in proteins. Since this replacement occurs in a random fashion, the selenium moiety does not appear to be involved in the specific biological function of such proteins (Sliwkowski, M. X. and Stadtman, T. C. (1985), “Incorporation and Distribution of Selenium into Thiolase from Clostridium kluyveri,” pp. 3140-3144).
Inadequate methodology is a major factor contributing to the lack of specific information regarding the nature of oxidized sulfur- or selenium-containing amino acid products. Precise, accurate analysis of sulfur- or selenium-containing amino acids in protein, e.g., cysteine, cystine, and methionine, and their corresponding oxidation products, requires considerable time and effort involving performic acid oxidation of the thiols and disulfides to cysteic acid (Hirs, C. H. W. (1967), “Performic acid oxidation,” Meth. Enzymol. 11:197-199; Manneberg, M. et al. (1995), “Quantification of cysteine residues following oxidation to cysteic acid in the presence of sodium azide,” Anal. Biochem. 231:349-353) and removal of any remaining oxidant and all water from the sample, followed by acid hydrolysis and chromatographic separation and quantification of cysteic acid (Hirs, C. H. W. (1967), “Performic acid oxidation,” Meth. Enzymol. 11:197-199).
U.S. Pat. No. 5,559,038 “Gas Chromatography/Mass Spectrometry Determination of Oxidized Sulfhydryl Amino Acids,” which is incorporated in its entirety by reference herein to the extent not inconsistent with the disclosure herewith and all references disclosed in the '038 patent are incorporated in their entirety by reference herein to the extent not inconsistent with the disclosure herewith, discloses a method for determination of in vivo concentration in a body fluid of the oxidized sulfhydryl amino acids cysteine sulfinic acid, cysteic acid, homocysteine sulfinic acid, and homocysteic acid. The method appears to involve combining an internal standard which is a deuterium labeled oxidized sulfhydryl amino acid noted above, with a body fluid containing an oxidized sulfhydryl amino acid; then at least partially purifying the oxidized sulfhydryl amino acid and the internal standard from other components of the body fluid; quantifying the oxidized sulfhydryl amino acid concentrations by gas chromatography/mass spectrometry and correcting for losses in oxidized sulfhydryl amino acid by determining losses in the deuterium labeled oxidized sulfhydryl amino acid. The method disclosed in the '038 patent appears to be quantifying unknown metabolites containing oxidized sulfhydryl amino acids. The oxidized sulfhydryl amino acid are not measured as concentrations in proteins, but rather as concentration in a body fluid and thus the proteins are not hydrolyzed to their constituent amino acid residues prior to analysis. Furthermore, the method uses gas chromatography/mass spectrometry to quantify the oxidized sulfhydryl amino acids.
Currently, there are no fully validated markers of oxidative stress in human populations. Biomarkers of oxidative stress are needed, among other reasons, to identify etiological relationships, to further define the pathophysiological mechanisms underlying diseases related to inflammation and oxidative stress, and to aid in assessing the efficacy of environmental, nutritional and therapeutic interventions.